title: INI1/hSNF5-interaction defective HIV-1 IN mutants exhibit impaired particle morphology, reverse transcription and integration in vivo.
abstract: BACKGROUND
  Retroviral integrase catalyzes integration of viral DNA into the host genome. Integrase interactor (INI)1/hSNF5 is a host factor that binds to HIV-1 IN within the context of Gag-Pol and is specifically incorporated into HIV-1 virions during assembly. Previous studies have indicated that INI1/hSNF5 is required for late events in vivo and for integration in vitro. To determine the effects of disrupting the IN-INI1 interaction on the assembly and infectivity of HIV-1 particles, we isolated mutants of IN that are defective for binding to INI1/hSNF5 and tested their effects on HIV-1 replication.
  
  
  RESULTS
  A reverse yeast two-hybrid system was used to identify INI1-interaction defective IN mutants (IID-IN). Since protein-protein interactions depend on the surface residues, the IID-IN mutants that showed high surface accessibility on IN crystal structures (K71R, K111E, Q137R, D202G, and S147G) were selected for further study. In vitro interaction studies demonstrated that IID-IN mutants exhibit variable degrees of interaction with INI1. The mutations were engineered into HIV-1(NL4-3) and HIV-Luc viruses and tested for their effects on virus replication. HIV-1 harboring IID-IN mutations were defective for replication in both multi- and single-round infection assays. The infectivity defects were correlated to the degree of INI1 interaction of the IID-IN mutants. Highly defective IID-IN mutants were blocked at early and late reverse transcription, whereas partially defective IID-IN mutants proceeded through reverse transcription and nuclear localization, but were partially impaired for integration. Electron microscopic analysis of mutant particles indicated that highly interaction-defective IID-IN mutants produced morphologically aberrant virions, whereas the partially defective mutants produced normal virions. All of the IID-IN mutant particles exhibited normal capsid stability and reverse transcriptase activity in vitro.
  
  
  CONCLUSIONS
  Our results demonstrate that a severe defect in IN-INI1 interaction is associated with production of defective particles and a subsequent defect in post-entry events. A partial defect in IN-INI1 interaction leads to production of normal virions that are partially impaired for early events including integration. Our studies suggest that proper interaction of INI1 with IN within Gag-Pol is necessary for proper HIV-1 morphogenesis and integration.
authors.affiliation: Department of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Sheeba
authors.initials: S
authors.lastname: Mathew
authors.forename: Minh
authors.initials: M
authors.lastname: Nguyen
authors.forename: Xuhong
authors.initials: X
authors.lastname: Wu
authors.forename: Achintya
authors.initials: A
authors.lastname: Pal
authors.forename: Vaibhav B
authors.initials: VB
authors.lastname: Shah
authors.forename: Vinayaka R
authors.initials: VR
authors.lastname: Prasad
authors.forename: Christopher
authors.initials: C
authors.lastname: Aiken
authors.forename: Ganjam V
authors.initials: GV
authors.lastname: Kalpana
mesh_terms: D002460:Cell Line; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019427:HIV Integrase / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D054884:Host-Pathogen Interactions; D006801:Humans; D046529:Microscopy, Electron, Transmission; D048348:Reverse Transcription / Q000502:physiology*; D000071796:SMARCB1 Protein; D014157:Transcription Factors / Q000378:metabolism*; D014771:Virion / Q000648:ultrastructure; D019065:Virus Assembly / Q000502:physiology*; D016662:Virus Integration / Q000502:physiology*
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D000071796:SMARCB1 Protein; C513266:SMARCB1 protein, human; D014157:Transcription Factors; D019427:HIV Integrase